Over 50% of the body's vitamin B6 goes for the function of the glycogen phosphorylase enzyme. Yet, its mechanism is perhaps the least known of all the vitamin B6 reactions. There are presently two heavily debated mechanisms. Quite interestingly, unlike other B6 reaction mechanism, neither of these involve the mobile conjugated electronic system of the bound pyridoxal-5'-phosphate (PLP) confactor. One proposal considers that the anionic 5'-PO4 in the active form functions as a reversible proton donor (Bronsted acid) in the glycosyl transfer of the reaction. The other proposal considers that the 5'-PO4 suffers a nucleophilic attach (a Lewis acid) by the glucose phosphate. One major difference in the proposal is the required charge on the 5'-PO4. The electronic excitation of the PLP cofactor by a uv laser is the basis for a new technique for the study of vitamin B6 reactions. The transient state of the photoexcitation is quite sensitive to the charge on the cofactor. A titration study of a model Schiff base proved that a more negative charge at the 5'-PO4 produced a faster decay of the transient. In the enzyme the results agree with those 31P NMR experiments which substituted thio or arsenate analogs for interfering phosphates. An advantage with the laser technique is that phosphates do not interfere. Quite interestingly the experiment with glucose-l-P shows for 5'-PO4 a charge slightly more positive than the monoaninon; a charge which is in opposition to one proposal. The research herein proposes to determine why and how the monoanionic 5'-PO4 in the enzyme acquires this charge. The cofactor will be replaced with analogs which will vary the charge. Excitation of the reconstituted enzyme should indicate the nature of the charge effect required for catalysis. A neutral species at the 5 feet position, e.g. pyridoxal, may indicate charge effects by the activated protein at this site. Substrate analogs which vary the PO4-PO4 interactions at the active site may demonstrate the ability of the interactions in triggering the negative charge reduction of the 5'-PO4. The why is more difficult to answer. Does the monoanion function in both directions of the phosphorylase reaction? This question should be approached with ternary complexes of holoenzyme, Pi or glucose-1-P, and glycogen.